| GDP-L-fucose(guanosine 5′-diphosphate(GDP)-L-fucose),as an important nucleotide sugar,is an essential glycosyl donor in the fucosylation of glycan,proteins and other biomacromolecules in organisms.In addition,GDP-L-fucose is an important intermediate for the synthesis of human milk oligosaccharides(HMOs),and plays a key role in the biological process of synthesizing fucosylated HMOs through microbial cell factories.Therefore,the use of metabolic engineering strategies to construct recombinant strains with high-efficiency synthesis ability of GDP-L-fucose is significant for the production of fucosylated compounds,especially fucosylated HMOs.In this study,metabolic engineering strategies were used for de novo synthesis of GDP-L-fucose using glucose as sole carbon source in engineered E.coli,and high titer and high conversion rate production of GDP-L-fucose were achieved through fedbatch fermentation.The main findings are as follows:(1)Recombinant plasmids p ET-gmd-wca G and p CDF-man C-man B were constructed and transformed into E.coli BL21(DE3),and four key enzymes of GDP-L-fucose de novo synthesis pathway Man B,Man C,Gmd and Wca G was overexpressed in engineered E.coli BL21(DE3).The recombinant strain was cultured in DM medium and induced by IPTG for 20 h.The shake flask fermentation results showed that the titer of GDP-L-fucose was 2.0 mg/L.In order to balance the metabolic flux of the synthetic pathway,four key enzymes of the metabolic pathway were divided into upstream module(synthesis of GDP-D-mannose,including enzymes Man B and Man C)and downstream module(synthesis of GDP-L-fucose,including enzymes Gmd and Wca G),respectively,to adjust the expression intensity of the two modules.By reducing the plasmid copy number of upstream module(p ACYC-man C-man B)and increasing the plasmid copy number of downstream module(p ET-gmd-wca G),the GDP-L-fucose titer of the engineered strain reached 5.3 mg/L,a 1.6-fold increase compared with that obtained by the initial strain.(2)In order to further regulate the expression intensity of the two modules,the strength of the ribosome binding site(RBS)was optimized.By using a combination of three different strengths of RBS(including high,medium,and low strength),the protein translation strength of target genes was regulated.The results showed that when the translation intensity of upstream module was relatively low and the translation intensity of downstream module was relatively high,the GDP-L-fucose production of recombinant strain EWL31 was further increased,reaching 6.8 mg/L,which was 3.4-fold compared with the initial production.(3)Considering that GDP-L-fucose is the precursor for the synthesis of colanic acid,this competitive metabolic pathway can be blocked by knocking out the endogenous gene wca J in E.coli.Fermentation results showed that inactivation of wca J significantly increased the intracellular accumulation of GDP-L-fucose.The GDP-L-fucose titer of recombinant strain EWL34 was 12.3 mg/L,which was 6.1-fold compared with the initial poduction.(4)The enzymes Man C and Wca G in the metabolic pathway require the cofactor guanosine5′-triphosphate(GTP)and the coenzyme NADPH,respectively,to participate in the enzyme reaction.Therefore,by cloning and overexpressing the gene zwf encoding glucose 6-phosphate dehydrogenase and the gene gsk encoding guanosine-inosine kinase,the regeneration of intracellular coenzyme NADPH and cofactor GTP was enhanced,thereby further increasing GDP-L-fucose production.The production of GDP-L-fucose in shake flask fermentation of recombinant strain EWL37 was further increased to 18.3 mg/L,which was 8.1-fold compared with the initial production.(5)In order to further increase the production of GDP-L-fucose,fed-batch cultivation of strain EWL37 was conducted.The fermentation results showed that after 40 h fed-batch fermentation in a 3 L fermentor,the intracellular accumulation of GDP-L-fucose reached 106mg/L,and the dry cell weight(DCW)reached 24.8 g/L.The specific GDP-L-fucose content was 4.3 mg/g DCW. |
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